Nanosyringe array and method

ABSTRACT

A nanosyringe is constructed using micro fabrication and nano fabrication techniques on a silicon substrate. The nanosyringe includes a membrane of silicon carbide. The position and operation of individual nanosyringes, arranged in an array of nanosyringes, can be independently controlled. A nanosyringe array can inject or extract a fluid from one or more cells or other structures. Microfluidic structures coupled to the nanosyringe allow external pumping or extraction. A cell matrix or organelles of individual cells can be non-destructively sampled in real time.

RELATED APPLICATION

This application is a divisional under 37 C.F.R. 1.53(b) of U.S. patentapplication Ser. No. 10/178,056 filed Jun. 21, 2002, which claimspriority under 35 U.S.C. 119(e) to U.S. Provisional Patent ApplicationSer. No. 60/300,013 filed on Jun. 21, 2001, which applications areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to nanosyringes, and in particular to ananosyringe array and method of making the nanosyringe array.

BACKGROUND

Microinjection techniques have been used for a variety of applications,including high-efficiency transformation, protein injection, pathogeninjection and organelle transfer. Transfer of DNA to mammalian cellcultures and embryos using microinjection has also been performed. Morerecently, microinjection has been employed to develop transgenic animalsfor pharmacological studies in the cardiovascular system, endocrinesystem, cancer and toxicology. It has also been used to examine the roleof the c-fos gene as a mitogenic signal in mammalian cells by injectionof protein inhibitors and monoclonal antibodies that block mitogenicactivities.

Micropipettes are primarily constructed of tapered borosilicate glass,quartz, or aluminosilicate needles with a minimum diameter of between 50and 100 nm. The primary disadvantages of these pipettes include inherentdamage inflicted on host cells, the inability to accurately controlinjection rates, the inability to inject more than one cell at a time,and the inability to inject more than one sample into a given cell atone time. Recently, a galinstan expansion femtosyringe was formed thatreduced the damage inflicted on host cells. In addition, heat inducedgalinstan (also known as gallinstan, a liquid metal alloy of gallium,indium and tin) was used to accurately control the rate of injection.These femtosyringes permit the injection of subcellular organelles suchas vacuoles, mitochondria and chloroplasts while maintaining theintegrity of cell membranes. Such femtosyringes are expensive to form,and do not facilitate the injection of more than one cell at a time, nordo they provide the ability to inject different substancessimultaneously into the same cell. Expensive needle puller equipment isalso required to form femtosyringes.

In one attempt to provide an array of microneedles, a plurality ofparallel hollow non-silicon microneedles are formed on a planar surfaceof a substrate. Multiple arrays of these needles can be coupled to forma three dimensional array with the substrates still attached, orremoved. Cross coupling channels provide for free fluid flow. The arrayis used to increase the flow rate of a fluid to be injected. Further,the size of the needles constructed using this technique are much largerthan those required to permit the injection of subcellular organelles,and may lead to unacceptable damage to cellular structures.

SUMMARY

A nanosyringe is constructed using micro and nano fabrication techniqueson a substrate. In one embodiment, the nanosyringe is formed as amembrane of silicon carbide or silicon nitride on a silicon substrateusing photolithography or other means. The nanosyringe comprises a tipfor penetrating a host without destroying the integrity of a hostmembrane. As used herein, the term needle is interchangeable with theterm syringe.

In one embodiment, an array of nanosyringes comprises a large number ofindependently controlled nanosyringes suitable for injecting a largenumber of cells or other structures at a given time, or injecting avariety of samples into a single cell at one time or at staggered timeintervals. In one embodiment, each nanosyringe is independentlycontrolled with respect to injection properties. The spacing of thenanosyringes is adjusted based upon a specific objective at the time offormation of the array. For example, arrays with a large spacing of 5-10μm may be used for injecting large numbers of cells. As another example,arrays with smaller spacing, such as less than 50 nm between tips, maybe used for injection various samples into a single cell at specificrates, time intervals and location. They are further used to increasethe flow rate of a sample to a cell. In one embodiment, a variety ofsamples can be injected in varying amounts and at varying times.

In one embodiment, the arrays are utilized to draw fluid or removesamples from cells. An external pumping system coupled to one or morenanosyringes allows non-destructive sampling of a cell matrix ororganelles of a cell as well as real time sampling and analysis ofphysiological changes within an individual cell. In one embodiment, ananosyringe both injects a first fluid and extracts a second fluidcoincident with a single penetration of a host membrane.

In one embodiment, sensor and detection capabilities, as well asmicro-pumps and valves are directly integrated into the system usingmicro and nano fabrication techniques on a semiconductor substrate. Thisprovides the ability to instantaneously sample a cell's cytoplasmfollowing the addition of a particular drug injected into the nucleus ofthat cell. Arrays of nanosyringes are also formed for a variety ofmicrofluidic systems where precise delivery of liquids is desired. Inone embodiment, a system is provided to independently positionindividual nanosyringes within a three axis coordinate system.

In one embodiment, a silicon carbide nanosyringe is constructed usingmicro and nano fabrication techniques on a silicon substrate. Eachnanosyringe is independently controlled with respect to injectionproperties. An external pump system coupled to a nanosyringe arrayallows non-destructive sampling of the cell's matrix and organelles, andreal time sampling and analysis of physiological changes withinindividual cells. Sensor and detection capabilities, as well asmicro-pumps and valves are directly integrated into the system usingmicro and nano fabrication techniques on a semiconductor substrate.

The present subject matter includes fabrication of thin, suspendedmembranes supported by a silicon substrate. In various embodiments, themembrane includes thin film materials such as silicon nitride or siliconcarbide. In one embodiment, the membrane is formed using a non-planar(that is, not flat) surface. The present subject matter includesmembranes formed using a cylinder, column or cone.

Other aspects of the invention will be apparent on reading the followingdetailed description of the invention and viewing the drawings that forma part thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like numerals describe substantially similar componentsthroughout the several views. Like numerals having different lettersuffixes represent different instances of substantially similarcomponents.

FIG. 1 illustrates a scanning electron microscope micrograph of an arrayof silicon tips used to form nanosyringes.

FIG. 2 illustrates a cross section of a nanosyringe.

FIG. 3 illustrates a perspective view of the nanosyringe of FIG. 1.

FIG. 4 illustrates a perspective partial section view of a self-alignednanosyringe.

FIG. 5 illustrates a perspective partial section view of an array ofself-aligned nanosyringes.

FIG. 6 illustrates a view of a positionable nanosyringe relative to asilicon substrate.

FIG. 7 depicts a method of fabricating a nanoneedle (or nanosyringe)array.

FIG. 8 illustrates a scanning electron micrograph of a Si₃N₄ needle.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings which form a part hereof, and in which is shown byway of illustration specific embodiments in which the invention may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the invention, and it is tobe understood that the embodiments may be combined, or that otherembodiments may be utilized and that structural, logical and electricalchanges may be made without departing from the spirit and scope of thepresent invention. The following detailed description is, therefore, notto be taken in a limiting sense, and the scope of the present inventionis defined by the appended claims and their equivalents. In thedrawings, like numerals describe substantially similar componentsthroughout the several views. Like numerals having different lettersuffixes represent different instances of substantially similarcomponents.

In various embodiments, the present subject relates to a stationarynanosyringe, an array of self-aligned stationary nanosyringes and anarray of individually positionable nanosyringes.

A method of making a nanosyringe or nanosyringe array includes formingat least one silicon tip as shown at 110 in FIG. 1. In one embodiment,the tips are atomically sharp. An array of silicon tips is shown inFIG. 1. The silicon tips 110 serve as a sacrificial material for syringefabrication. The tips 110 shown in FIG. 1 are less than approximately 10nm in diameter, and each has a shaft 120 which is approximately 1 μm indiameter. These sizes referred to are merely one example. Other sizes,as well as materials other than silicon, are well within the scope ofthe invention as will be apparent from a description of the processsteps used to form the tips.

Silicon wafer 130 is initially oxidized at 1100° C. in a steam ambientto form a layer of silicon dioxide. A pattern of dots approximately 500nm in diameter are defined through a lithographic process, such asphotolithography using an i-line stepper. The pattern is transferredonto the silicon dioxide layer through fluorine-based reactive ionetching (RIE). This is followed by chlorine-based inductively coupledplasma (ICP) RIE to transfer the pattern onto the silicon substrate,resulting in an array of dots.

The silicon layer is again oxidized. Localized stress effects actingaround the neck of the post or cylinder produces an atomically sharptip. Reactive ion etching is used again to remove the oxide from thefloor of the silicon layer. Then another chlorine RIE is performed tofurther etch the silicon and create the shaft 120 for each tip 110. Thesilicon dioxide is removed using a 1:6 buffered hydrofluoric acidsolution. Methods other than those described herein can be used towholly or partially remove the silicon substrate.

In one embodiment, a faceted profile or pedestal at the base of theshaft is formed by anisotropic wet etching. For example, using (100)silicon, an anisotropic wet etchant such as potassium hydroxide yields<111> cuts at an angle of 54.74° relative to the surface of the siliconsubstrate. The cut is illustrated in FIG. 2 at 210A, which shows a crosssection of a finished nanosyringe indicated generally at 200A.

The angle of the cut is determined, in part, by the orientation of thecrystal planes of the silicon substrate. The orientation of the crystalplanes are expressed using Miller indexes and relate to how the siliconcrystal is sliced. Wet anisotropic etching will etch the silicon atdifferent rates depending on the orientation of the crystal planes.

The membrane is next fabricated on the silicon tip, shaft and facetedbase followed, in one embodiment, by removal of the silicon structure bya combination of wet and dry etching steps. Selective removal of thesilicon can result in structural supporting members within or about thenanosyringe.

FIG. 3 provides a perspective of a nanosyringe. To obtain the finishednanosyringe, the previously formed silicon tip is coated with aconformal membrane material 220 such as silicon nitride or siliconcarbide. A portion of the silicon tip is removed through a combinationof wet and dry RIE etching to leave a core open area, or syringe cavity,230 for fluid flow. The syringe cavities are connected in one embodimentto channels and reservoirs for fluid dispensing. A base structuralsupport area 240 remains following the etching to provide support forthe syringe membrane material 220.

An opening, or nozzle, at the tip of the syringe 250 is formed using aprocess similar to submicron nozzle fabrication. In one embodiment, apolymer layer or other photoresist material is used as an etch mask. Thetip may be etched using traditional etching methods.

In one embodiment, a localized stress effect can be used to form asyringe nozzle. In this embodiment, a concentration of stressesoccurring within the membrane at a sharp radius portion of the siliconnitride causes a fracture which forms a nozzle on the syringe. Thesharp, or small, radius portion of the membrane separates from a largerradius portion.

In one embodiment, silicon carbide or other material is etched back toform a syringe nozzle. In one embodiment, greater uniformity in nozzleplacement and dimension is achieved by an etching process.

In one embodiment, low-pressure chemical vapor deposition (LPCVD) isused to deposit the membrane on the silicon tip. In one embodiment,LPCVD is used to deposit silicon nitride and localized stresses arecontrolled by introduction of controlled amounts of oxygen in the film(or membrane) during deposition, thus forming silicon oxynitride.

In general, LPCVD provides a membrane pin-hole free and with uniformphysical properties. Stresses induced are generally characterized astensile and the range of levels stress tends to be rather narrow.

In one embodiment, the process of membrane fabrication includes plasmaenhanced chemical vapor deposition (PECVD). As with LPCVD, the depositedfilm is determined by a chemical reaction between the source gasessupplied to the reactor. Since the resulting films arenon-stoichiometric, a wide range of stress values can be obtained, fromtensile stress to compressive.

In one embodiment, the silicon surface of the tips is converted tosilicon carbide (SiC) by carbonization. Silicon carbide conformallycoats the silicon tip and when carbonized, the membrane provides asharper and stronger nanosyringe.

In one embodiment, the nanosyringe remains stationary and a cell ismoved into position such as by using a microelectromechanical systems(MEMS) actuator driven stage. In one embodiment, the cell is positionedusing laser tweezers.

FIG. 4 shows nanosyringe 410 adapted for penetrating cell 420. A facetedcavity 430 is formed in the same manner as the faceted pedestal in FIG.2 using an anisotropic etch in place of the chlorine RIE to removesilicon to form the shaft of the syringe. Faceted cavity 430 is used totrap and position the cell at the same time. In one embodiment, cell 420is supported by flat quartz slide 430. Syringe 410 is self-alignedwithin faceted cavity 430. In addition to, or in lieu of, the facetedcavity, other structure may be formed in the silicon substrate tocapture cell 420. For example, conical structures or posts may be usedto position cell 420 for penetration by nanosyringe 410.

In one embodiment, quartz slide 430 comprises a cell culture, an arrayof syringes, each having a faceted cavity, is lowered against the cellculture to trap and hold cells in place in a position reachable by thesyringes as shown 510 in FIG. 5. In one embodiment, a channel for eachsyringe is provided in a backside of the substrate at 515A, 515B and515C. As previously indicated, each channel provides an independent pathto a reservoir. The paths may be connected in some embodiments so thatmore than one syringe is coupled to the same reservoir, or multiplechannels and syringes may have separate reservoirs. In furtherembodiments, more than one syringe is formed within a faceted cavity tofacilitate the injection of multiple samples or substrates into aparticular cell at a specific time or time intervals.

Patterning the backside of the silicon wafer includes leaving somesilicon to make channels, pumps, and other structures for coupling toother systems for fluid handling.

For example, in one embodiment, a fluid reservoir is patterned into thesilicon. The reservoir receives fluid by capillary extraction orcontains fluids for delivery using the nanosyringe.

FIG. 6 illustrates a positionable nanosyringe adapted for movementrelative to a silicon substrate. MEMS actuators (including electrostaticcomb drives or piezo elements, for example) drive the syringe laterallyin the x-axis and y-axis to precisely position the syringe with respectto the object to be injected.

In one embodiment, syringes and arrays are movable with respect to thecavity. Incorporation of a flexible structure or suspension mechanismfor syringe displacement and inclusion of actuators for moving thesyringe in a plane, as well as up and down, are utilized. In oneembodiment, electrostatic actuators for x-axis and y-axis displacementare constructed using the remaining backside of the silicon substrate.In one embodiment, the electrostatic actuator includes one or more combdrive actuators. Out-of-plane z-axis motion is provided by similaractuators by means of electrostatic levitation. In one embodiment,corrugated structure 600 is disposed concentric to the syringe and actsas a bellows to allow freedom of movement of the syringe.

In one embodiment, both independent steering and a self-alignmentfaceted cavity are used with a particular nanosyringe.

EXEMPLARY EMBODIMENT

The following describes one embodiment of the present subject matter.

Standard <100> silicon wafers is used as the base material for the arrayfabrication. The individual fabrication steps are depicted in FIG. 7.Initially the wafers were thermally oxidized at 1100° C. in a steamambient with trichlorethane (TCA) to form a 0.7-μm thick layer of SiO₂(FIG. 7A). The wafers were then primed at 90° C. in hexamethyldisilazane(HMDS) vapor to promote photoresist adhesion, followed by photoresistcoating. A photolithography step was performed using a 10X i-linestep-and-repeat system to form an array of 0.5 μm dots. The dot patternis then transferred onto the silicon dioxide layer by magnetron-assistedreactive ion etch, using CHF₃ (30 sccm) at 1 kW until the open areaswere free of SiO₂.

This patterned silicon dioxide (FIG. 7B) then serves as an etch mask toetch the underlying silicon to define standing silicon posts. The postswere etched using a chlorine-based inductively coupled plasma (Cl₂=50sccm, BCl₃=2.5 sccm, ICP power=75 W) to obtain 1-μm tall silicon posts(FIG. 7C). A second thermal oxidation step is performed to turn thesesilicon posts into atomically sharp tips. When the posts are thermallyoxidized, a stress effect around the base of the post causes an unevenoxidation along the length of the post thus resulting in a cone-likestructure. This was performed in a steam ambient, with TCA added, at1100° C., for about 30 minutes (FIG. 7D). The initial oxide whichremained on top of the tips can then be used as an etch mask to define ashaft at the base of the tips. To do this, a plasma etchback step isperformed in CHF₃ ambient to strip the oxide surrounding the tips. Thisis followed by a chlorine ICP etch step to define the shafts (FIG. 7E).The samples are then immersed in a 1:6 buffered hydrofluoric acidsolution to strip all the remaining oxide, thus exposing the tips (FIG.7F). FIG. 1 illustrates a typical silicon tip array obtained by thisprocess.

Subsequent steps form the needles. By way of overview, the needles areformed by a suspended silicon nitride membrane utilizing the tip arrayas a mold. Suspended silicon nitride membranes are formed into acorrugated membrane, which conforms to the shape of the tips. Thesamples are coated with a layer of low-stress Si₃N₄ using low-pressurechemical vapor deposition (LPCVD) as shown in FIG. 7G. The thickness ofthis layer depends on the needle diameter and aperture desired.Photolithography is performed on the backside of the wafers using aninfrared aligner. This defines the windows for the through-wafer etch.The wafers are then immersed in a 50% wt. potassium hydroxide solutionat 90° C. until the cores of the tips are removed.

The needle apertures are created using a process similar to submicronnozzle fabrication. In one embodiment, photoresist is spun to completelycover the needles and an RIE etchback is performed. FIG. 8 shows acompleted needle.

ADDITIONAL EMBODIMENTS

In one embodiment, materials other than silicon and silicon carbide areused for fabrication of a nanosyringe. For example, thin metal filmsdeposited using techniques such as, but not limited to, chemical vapordeposition, physical vapor deposition, electroplating, and electrolessplating.

In one embodiment, a nanofabricated array of syringes is adapted fortranscutaneous injection of medicament or for drawing blood or cellsamples. In such an embodiment, a plurality of nanosyringes, eachfabricated of silicon carbide, are arranged on a substrate havingstructural silicon reinforcement beams or members. The depth ofpenetration of a nanofabricated array of syringes may be sufficientlyshallow to avoid disturbing nerve cells and therefore offers a low painmethod of drawing samples or delivering medicine. The present subjectmatter may be used to inject fluids into blood cells or to extract cellmatrix or organelles for further analysis.

In one embodiment, the nanosyringe structure is strengthened byproviding reinforcing members on the silicon substrate. For example, inone embodiment, the silicon includes a network or grid of reinforcementbeams etched or otherwise formed into the backside of the array orwithin the nanosyringe. In one embodiment, the nanosyringe is fabricatedof a membrane material suited for a harsh environment.

In one embodiment, the methods and devices described herein are appliedto the fabrication and use of syringes that are larger or smaller thannano scale dimensions. For example, in various embodiments, the syringesare more properly described as millisyringes, microsyringes,picosyringes or femtosyringes.

In one embodiment, one or more microfluidic devices or actuators arecoupled to a nanosyringe. For example, in one embodiment, a flow valveis coupled to a nanosyringe. Other microfluidic devices are alsocontemplated, including, but not limited to pumps, reservoirs, sensors,fluid conduits or channels. In one embodiment, a microfluidic device oractuator is fabricated on the same silicon substrate as the nanosyringe.In one embodiment, the nanosyringe is fabricated on a first siliconsubstrate and a microfluidic device or actuator is fabricated on asecond silicon substrate and the first and second substrate aresubsequently bonded together.

In one embodiment, the membrane is fabricated of material other thansilicon carbide. Silicon carbide exhibits robust performance in a harshenvironment, has good mechanical hardness and is relatively chemicallyinert. Polycrystalline SiC can be deposited using PECVD. Also, 3C-SiCcan be formed on silicon by carbonization of the silicon surface. Ineither case, thin conformal films can be directly deposited on silicon.Silicon can be used as a sacrificial member because of the high chemicalselectivity between Si and SiC. The SiC film can be released by etchingusing wet chemistries such as potassium hydroxide,ethylene-diamine/pyrocatechol (EDP) or hydrofluoric acid. Highselectivity is also found in reactive ion etching which also allowsfabrication of mechanical supporting structures within thenanofabricated device.

In one embodiment, the nanosyringe or nanosyringe array is opticallytransparent. Transparency allows monitoring operation of the nanosyringevia an optical microscope.

In one embodiment, the tip is formed on the silicon substrate without acylinder or shaft at the base. The membrane is formed on the tip asdescribed above. In one embodiment, microfluidic devices or MEMS devicesare coupled to the nanosyringe, also as described above.

CONCLUSION

The above description is intended to be illustrative, and notrestrictive. Many other embodiments will be apparent to those of skillin the art upon reviewing the above description.

1. A method comprising: forming a column on a silicon substrate, thecolumn having an atomically sharp tip; depositing a membrane on thecolumn; removing a portion of the column after depositing the membrane;and etching the membrane to form a nozzle end.
 2. A device comprising: amembrane formed by depositing a conformal layer of silicon nitride on asilicon post oriented perpendicular to a silicon substrate, the membranehaving an orifice; and a fluid reservoir in fluidic communication withthe orifice.
 3. The device of claim 2 further comprising a cellretention cavity.
 4. A method comprising: transferring a pattern of dotsonto a silicon substrate; oxidizing the substrate to form an atomicallysharp silicon tip corresponding to each dot; creating a shaft for eachsilicon tip; forming a pedestal at the base of each shaft; coating thesilicon tips with a membrane material; and removing a portion of thesilicon from each tip.
 5. The method of claim 4 further comprisingforming an opening in each tip.